Method for producing human antibodies to a self physiological receptor by site-directed in vitro immunization of human lymphocytes

Abstract

A method for obtaining agonist, antagonist and inverse agonist, to a given physiological receptor is disclosed. For the method, use is made of in silico design synthetic immunogens, which are caused to act in vitro on human lymphocyte-containing cell populations. A preferred receptor is human CD152, particularly the regions of CDR1, CDR2 and CDR3 that elicit antibodies serving as antagonist, inverse agonist and agonist, respectively. Also provided is a method in the treatment of human peripheral lymphocytes for use in the screening of CD152 ligands that yield pharmacological effects.

1. A method for preparing human antibodies recognizing a self physiological receptor, comprising following steps:
 (a) providing a group of lymphocytes obtained from a naïve human donor; 
 (b) depleting CD56 +  lymphocytes; 
 (c) culturing said lymphocytes with in silico designed synthetic antigens encompassing amino acid sequence of self physiological receptor in vitro in the presence of CD40L; 
 (d) adding Epstein-Barr virus (EBV) to the immunized lymphocytes; and 
 (e) identifying EBV-infected cells that produce the antibody that recognizes the receptor. 
 
     
     
       2. The method of  claim 1 , wherein the resultant human antibodies are collected and screened for the presence of pharmacologic functions. 
     
     
       3. The method of  claim 1 , wherein the antigens are peptides comprising one or more Complementarity Determining Regions (CDRs) of Cytotoxic T-Lymphocyte Antigen 4(CD152), or an immunogenic fragment thereof. 
     
     
       4. The method of  claim 3 , wherein the antigens are peptides comprising one or more sequences selected from the group consisting of SEQ ID NOS: 2-4. 
     
     
       5. The method of  claim 3 , wherein the antigens are peptides comprising one CDR of CD152, or an immunogenic fragment thereof, and SEQ ID NO: 1. 
     
     
       6. The method of  claim 3 , wherein the antigens are peptides comprising one or more sequences selected from the group consisting of SEQ ID NOS: 5-7.